August 28, 2019 -- Concerns arising over the development of hypervirulent strains of Klebsiella pneumoniae have led researchers to develop an entirely new vaccine to protect against the gram-negative bacteria. A team of researchers at Washington University School of Medicine in St. Louis and VaxNewMo (a startup based in St. Louis) designed the vaccine by genetically manipulating E. coli. The details of the prototype designed were published in the Proceedings of the National Academy of Sciences on August 27th.
Klebsiella pneumoniae (Kp) causes a variety of healthcare-associated infections, including pneumonia, bloodstream infections, wound and surgical site infections, and meningitis, according to the Centers for Disease Control and Prevention. Moreover, these bacteria are becoming increasingly resistant to antimicrobials. Hypervirulent Klebsiella (hvKp) strains are now emerging via the acquisition of hypervirulence plasmids by multidrug resistant classic Klebsiella (cKp) strains. Last year, hypervirulent strains, primarily K1 and K2, of the bacteria caused tens of thousands of infections across China, Taiwan, and South Korea. These infections were responsible for nervous system damage and endophthalmitis within the victims.
Researchers set out to describe the recombinant production and analytical characterization of rapidly produced bioconjugate vaccines against the 2 most common hypervirulent serotypes of Kp (K1 and K2), which account for more than 70% of the hvKp cases. The vaccine was developed as a glycoconjugate composed of sugars linked to proteins. The researchers cloned the K1 and K2 capsular polysaccharide (CPS) loci from Kp. Next, the plasmids containing the biosynthesis machinery for the K1 or K2 CPS were introduced into E. coli CLM37, a reporter strain for heterologous polysaccharide expression and assembly. Lastly, the rmpA, a transcriptional activator, was also cloned from Kp to enhance the expression of K1 and K2 and introduced this plasmid into the modified E. coli.
Western blot analysis using the monoclonal antibody 4C5, specific to the K1 CPS of Kp, reacted with the modified E. coli was coexpressed the K1 CPS locus and RmpA, indicating that the polysaccharide produced by this glycoengineered E. coli strain has a K1 structure.
Western blotting: a method for identifying specific proteins from a cell’s mixture of proteins. This technique requires separating proteins based on molecular weight and size through gel electrophoresis and then transferring them to a membrane which bands each type of protein so that the protein of interest can be seen more clearly when labeled with corresponding antibodies.
The vaccine was tested in mouse models where they gave groups of 20 mice three doses of the vaccine or a placebo at two-week intervals. Then they challenged the mice with about 50 bacteria of either the K1 or the K2 type. Previous studies have shown that just 50 hypervirulent Klebsiella bacteria are enough to kill a mouse. Among the vaccinated mice, 80 percent infected with K1 and all of those infected with K2 survived.
"Glycoconjugate vaccines are among the most effective, but traditionally they've involved a lot of chemical synthesis, which is slow and expensive," said Christian Harding, senior author and co-founder of VaxNewMo. "We've replaced chemistry with biology by engineering E. coli to do all the synthesis for us." The researchers developed the first successful case of a vaccine providing protection from extremely lethal hypervirulent isolates.
"We are very happy with how effective this vaccine was," said first author, Mario Feldman. "We're working on scaling up production and optimizing the protocol so we can be ready to take the vaccine into clinical trials soon."
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